Toni K. Traeger

The pyruvate dehydrogenase complex (PDHc) ¹ links glycolysis to the Krebs cycle by catalyzing the oxidative decarboxylation of pyruvate to acetyl-CoA and CO₂, a process essential for life ^2,3 . PDHc is formed by structural proteins (E3-binding protein, E3BP) ^4–7 , enzymatic subunits (E1, E2, E3) ^4,6 , and mobile lipoyl domains (LDs), the latter shuttling intermediates across active sites ^6,8 . Although numerous details regarding pyruvate oxidation steps have been elucidated ⁹ , the precise organization of the entire PDHc remains unknown due to its large size and dynamic heterogeneity. Here, we employ in silico , in vitro , and in situ methods to propose a multi-scale model of PDHc that includes approximately one million atoms and to visualize multiple conformational states. This model reveals a ∼40-50 nm nested shell structure, formed by flexible linkers that spatially coordinate the E1 and E3 enzyme complexes around the E2-E3BP core scaffold. This structure acts as a molecular sieve, selectively guiding lipoyl arms while maintaining enzyme positioning with sub-nm precision. During catalysis, the nested shell structure expands and adopts a mechanically reinforced state comparable in magnitude to viral assemblies ¹⁰ . Our findings provide structural context for the textbook “link reaction” ¹¹ , building on decades of biochemical knowledge, are transferable to functional aspects of other α-ketoacid dehydrogenase complexes, and, ultimately, expand our understanding of primary metabolism as a whole. Abstract Figure:

Myo-inositol-1-phosphate synthase (MIPS) catalyzes the NAD ⁺ -dependent isomerization of glucose-6-phosphate (G6P) into inositol-1-phosphate (IMP), controlling the rate-limiting step of the inositol pathway. Previous structural studies focused on the detailed molecular mechanism, neglecting large-scale conformational changes that drive the function of this 240 kDa homotetrameric complex. In this study, we identified the active, endogenous MIPS in cell extracts from the thermophilic fungus Thermochaetoides thermophila . By resolving the native structure at 2.48 Å (FSC = 0.143), we revealed a fully populated active site. Utilizing 3D variability analysis, we uncovered conformational states of MIPS, enabling us to directly visualize an order-to-disorder transition at its catalytic center. An acyclic intermediate of G6P occupied the active site in two out of the three conformational states, indicating a catalytic mechanism where electrostatic stabilization of high-energy intermediates plays a crucial role. Examination of all isomerases with known structures revealed similar fluctuations in secondary structure within their active sites. Based on these findings, we established a conformational selection model that governs substrate binding and eventually inositol availability. In particular, the ground state of MIPS demonstrates structural configurations regardless of substrate binding, a pattern observed across various isomerases. These findings contribute to the understanding of MIPS structure-based function, serving as a template for future studies targeting regulation and potential therapeutic applications.

EMPIAR, the Electron Microscopy Public Image Archive centered at EMBL-EBI, is a public resource for raw electron microscopy images related to EMDB, contains micrographs, particle sets and tilt-series.

Cell-free systems display tremendous potential for biotechnological applications, complementing in vitro reconstituted enzymatic processes and traditional expression systems. However, they often represent “black boxes” without much insight into their components. Here, we characterize a thermophilic cell-free system that produces succinyl-CoA and discern its intrinsic, non-stochastic organization. By employing biochemical, biophysical, and bioinformatic methods we resolve its molecular composition, 3D architecture and molecular function at atomic resolution. We further report the high-resolution cryo-EM structure of the reaction’s main component, the oxoglutarate dehydrogenase complex core (E2o), which displays various structural adaptations. These include hydrogen bonding patterns confining interactions of participating enzymes (E1o-E2o-E3), electrostatic tunneling that drives inter-communication between subunits, and the presence of a flexible subunit, the E3BPo connecting E2o and E3. This multi-scale analysis of a cell-free system provides a blueprint for structure-function studies of complex mixtures of biotechnological value.